Project Summary/Abstract The interior and exterior of cells are delineated by a mostly impermeable barrier called the cell membrane. Proteins called membrane transporters enable the transport of molecules across this barrier to fulfill cellular needs such as the export of toxic substances or the import of essential nutrients. Many human diseases are associated with defects in the transport mechanisms of membrane transport proteins. The proposed research focuses on the SLC4 membrane transporter family, which includes a human protein called AE1 that is essential for driving cellular respiration. AE1 is the most abundant membrane protein in red blood cells, and modulates CO2 transport by exchanging bicarbonate and chloride ions. Diseases such as renal tubular acidosis, hereditary spherocytosis, and hereditary stomatocytosis can be caused by mutations in the membrane transport domain of AE1. We study yeast and plant homologs of human AE1 because they provide experimental advantages such as the ability to test their function through a simple but powerful genetic complementation assay. Our long-term goal is to study these homologs to elucidate the molecular mechanism of SLC4 transport activity and thus shed light on the molecular basis of these diseases and set the stage for the development of treatments. Yeast and plants each have SLC4 proteins that transport borate, which is chemically similar to bicarbonate. In yeast, the transport activity of the SLC4 protein Bor1 is to expel borate, which is toxic in excess. In plants, borate is an essential micronutrient that must be imported into plants to support their cell walls, but exported when in toxic levels of excess. The primary goal of the proposed research is to dissect the mechanism of transport in SLC4 borate transporters. We will do so through a combination of mutagenesis and functional assays, informed by our knowledge of structures of Bor1 and AE1. These experiments will identify and map amino acids that play functional roles in transport, determine how and whether the quaternary structure of SLC4 proteins controls transport activity, and dissect how the substrate binding affinity can explain distinct functional roles for different borate transporters.